Isolation and efficient expression of a specific monoclonal antibody is an essential aspect of modern bioscience, from basic research to development of human therapeutics. There are two methods of isolating a specific monoclonal antibody—the first is hybridoma technology (Kohler, G., and Milstein, C., Nature 256, 295-497 (1975) (this and all other references cited herein are hereby incorporated in their entirety herein)) and the second is display technologies using phage, bacteria and yeast (Sergeeva et al., 2006; Winter, G. et al., Annu Rev Immunol 12, 433-455 (1994)). Both technologies have their advantages and disadvantages.
Hybridoma technology consists of three main steps: 1) injecting an animal (typically a mouse) with an immunogen to trigger development of B cells producing various antibodies against the immunogen, 2) extracting the animal's B cells and fusing them with an immortal myeloma line (such as Sp2/0-Ag14 or NS0) resulting in a library of immortal, antibody-producing cells called hybridomas, and 3) identifying and isolating those hybridomas from the library that produce monoclonal antibodies with desired binding affinity and/or biological activities, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-mediated phagocytosis, apoptosis, cell growth inhibition, cell growth stimulation, and viral neutralization. The advantage of hybridoma technology is that each hybridoma cell line secretes its unique antibody into the growth medium of the cell line, making analyses of the antibody's biological activities relatively easy. The disadvantage of hybridoma technology is that it is not always possible for an immunized mouse to raise high affinity antibodies against certain human antigens or epitopes that are highly conserved between humans and rodents; the mouse's immune system does not recognize such a conserved antigen as “foreign” and does not produce antibodies against it. This is a phenomenon called tolerance (Rajewsky, K., Nature 381, 751-758 (1996)). To overcome this problem, non-rodent animals such as rabbits and chickens have been immunized to produce B cells for fusion (Nishinaka, S., et al., J Vet Med Sci 58, 1053-1056 (1996); Spieker-Polet, H., at al., Gene 344, 1-20 (1995)). However, B cells from rabbits and chickens do not generate immortal hybridomas efficiently, and moreover their resulting hybridomas are generally unstable in antibody production.
An alternative to hybridoma technologies is various display technologies using phage, bacteria cells and yeast cells (Sergeeva, A., et al., Adv Drug Deliv Rev 58, 1622-1654 (2006); Winter, G., et al., supra (1994)). In these display technologies, antibody fragments, typically Fab or single-chain Fv (scFv), are expressed on the surface of phage or cells. The phage particles (or cells) are then selected based upon binding affinity to an antigen of interest, and the genes encoding these antibody fragments are recovered from selected phage (or cells). The advantage of display technologies is the ability to create a large antibody fragment library from any species, as long as their variable region sequences are known. The library can be screened to select particular phage particles (or cells) exhibiting antibody fragments with desired antigen binding characteristics on the surface by the use of immobilized antigens. A disadvantage of these display technologies is that the antibody fragments of interest must be converted to the form of intact antibody molecules and expressed in a mammalian expression system to fully characterize their biochemical properties and biological functions, such as binding affinity, ADCC, CDC, antibody-mediated phagocytosis, apoptosis, cell growth inhibition, cell growth stimulation, and viral neutralization. To solve this problem, display technology has recently been used with mammalian cells to enable the isolation (Akamatsu, Y., et al., J Immunol Methods 327, 40-52 (2007)) and affinity maturation (Ho, M., et al., Proc Natl Acad Sci USA 103, 9637-9642 (2006)) of monoclonal antibodies. In these mammalian cell display systems, human IgG molecules attached to a glycosyl-phosphatidylinositol (GPI) anchor (Akamatsu, Y., et al., supra (2007)) or human scFv fragments fused to the transmembrane domain of a PDGF receptor (Ho, M., et al., supra (2007)) are expressed on the surface of mammalian cells. The advantage of using mammalian cells is that antibody molecules are expressed without folding and post-translational modification problems associated with non-mammalian cells. After selecting cells expressing membrane-bound antibodies with desired antigen binding properties, the genes of these membrane-bound antibodies are recovered from cells and modified to express a secreted form of the antibody. The modified genes are then reintroduced to mammalian cells, and the antibodies secreted into the culture medium of these cells lines can be analyzed for desired biological characteristics.
Thus, currently used display technologies can produce large antibody libraries from a wide range of species from which particular antibodies having desired antigen binding characteristics can be selected, but the genes encoding these selected modified antibodies have to be laboriously manipulated in order to produce intact soluble antibody molecules for analyses of their biological functions. Hybridoma technologies, on the other hand, readily yield soluble antibodies, but are only applicable to a limited number of species. Furthermore, since hybridomas are not physically linked to their secreted antibodies, they cannot be selected by the use of immobilized antigens to which the secreted antibodies may bind. Each hybridoma has to be grown individually, which often requires multiple rounds of subcloning, for detailed analyses of its secreted antibodies.
Therefore, in order to combine the advantages and eliminate the disadvantages of both display and hybridoma technologies, expression of whole antibody molecules by mammalian cells simultaneously in both their membrane-bound and secreted forms is desired. Particularly, it is ideal to express such secreted antibody in its fully intact soluble form. Cells can be selected based on antigen binding of their membrane-bound antibodies. Antibodies secreted in the culture medium of each of the selected cells can be tested for antigen-binding and biological activity without further manipulation of the cell and antibody-encoding genes.
Alternative RNA processing is a common strategy used by eukaryotes to produce more than one mRNA, resulting in more than one kind of polypeptide, from a single transcription unit (Smith, C. W., and Valcarcel, J., Trends Biochem Sci 25, 381-388 (2000); Stamm, S., et al., Gene 344, 1-20 (2005)). The gene coding for the human immunoglobulin mu heavy chain is one such gene (Peterson, M. L., Immunol Res 37, 33-46 (2007)). B lymphocytes produce two distinct forms of IgM molecules during differentiation—the monomeric, membrane-bound form in early-stage B cells and the pentameric, secreted form in terminally differentiated plasma cells. The switch between the synthesis of the two forms of IgM molecules is accomplished by alternative RNA processing of mu heavy-chain precursor RNA. The two forms of mu mRNA differ only in their 3′ termini (FIG. 1). Specifically, when the precursor mu RNA is cleaved and polyadenylated using the first poly(A) site located downstream of the CH4 exon (shown as “pA-s” in FIG. 1), the resulting mature mRNA produces the secreted form (S-form) of the mu heavy chain. Alternatively, when the first poly(A) site (pA-s) is removed by splicing between the CH4 and M1 exons, and the second poly(A) site (shown as “pA-m” in FIG. 1) is used, the resulting mRNA produces the membrane-bound form (M-form) of the mu heavy chain. When expressed, the two forms differ in their amino acid sequences at the C-terminal of their respective molecules. Although the molecular mechanism that controls the alternative processing of mu heavy chain mRNA is not fully understood to date (Borghesi and Milcarek, 2006; Peterson, M. L., supra (2007)), delicate balancing between two mutually exclusive RNA processing events, i.e., splicing of precursor mu RNA between the CH4 and M1 exons and its cleavage/polyadenylation at the first poly(A) site (pA-s), seems to be the key for determining the ratio between M-form and S-form of mu mRNA.
By incorporating the gene structure that mimics the 3′ region of the mu gene (hereinafter, a “Ig mu gene 3′ region mimetic”), which enables generation of two forms of mRNA from a single transcription unit by alternative processing of the common precursor RNA, and adjusting the balance between the two mutually exclusive RNA processing events in such constructed gene, we developed an expression vector capable of simultaneous expressing membrane-bound and secreted forms of a polypeptide in a single eukaryotic cell.